PT2016198E - Method for predicting a risk of adverse drug reactions by determination of hla-b 1502 - Google Patents

Abstract

This invention relates to a method of determining the presence of certain HLA alleles, such as HLA-B*1502 or HLA-B*5801, and a kit for carrying out this method. Also disclosed is a method for assessing whether a patient is at risk for developing adverse drug reactions (e.g., Stevens-Johnson syndrome, toxic epidermal necrolysis, or hypersensitivity syndrome) based on the presence or absence of a genetic marker (e.g., HLA-B*1502, HLA-B*5801, or HLA-B*4601).

Description

ΕΡ2016198Β1

METHOD FOR PREDICTION OF A RISK OF ADVERSE REACTIONS TO DRUGS BY MEANS OF THE DETERMINATION OF HLA-B 1502

BACKGROUND

An adverse drug reaction (RAF) is an undesired and unintended effect of a drug.

In particular, an adverse drug reaction occurs at the doses used for prophylaxis, diagnosis or therapy. According to a widely cited meta-analysis, RAF were classified as the fourth and sixth most common cause of death (Lazarou et al., JAMA, 279 (15): 1200-1205, 1998). Cutaneous RAF accounts for approximately 2-3% of all hospital admissions (Bigby et al., JAMA, 256 (24): 3358-3363, 1986). They range from mild maculopapular (MPE), with increasing severity, to life-threatening RAFs, such as hypersensitivity syndrome (SHS), Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (NET; Lyell). The mortality rate of the latter can be as high as 40%. SHS is characterized by the involvement of multiple organs (eg, hepatitis or nephritis) accompanied with systemic manifestations (eg, fever, arthroscopy, eosinophilia and lymphadenopathy) in addition to skin cracks (Roujeau et al., N. Engl. Med., 331: 1272-1285, 1994). SSJ and NET are hypersensitivity immuno-complex mediated disorders characterized by the rapid development of purpura blotting rash and target-type lesions accompanied with muscle involvement and skin detachments (Roujeau et al., J, Invest, Dermatol, 1994, 102: 218S-30S). They are mainly 1 ΕΡ2016198Β1 ΕΡ2016198Β1 caused by anticonvulsants, non-steroidal, Engl. J. Med., Phenobarbital anticonvulsants) and drugs, such as sulfonamides, allopurinol, anti-inflammatory and antimalarial drugs (Roujeau et al., N. 333 (24): 1600-1607, 1995). In Taiwan, (for example, CBZ, phenytoin and allopurinol are the most common drugs that cause SSJ / NET.

Recent developments in pharmacogenomics have implicated that susceptibility to RAF is associated with particular genetic alleles. For example, genomic polymorphisms of thiopurine methyltransferase genes are closely related to RAF induced by azathioprine, a drug for rheumatic diseases or cancer (Yates et al., Ann. Intern. Med., 126 (8): 608-614, 1997). It has also been suggested that susceptibility to SSJ / NET / SHS induced by certain drugs is genetically determined (Gennis MA, Am. J. Med., 91 (6): 631-634, 1991; Edwards SG, Postgrad Med. , 75 (889): 680-681, 1999). However, the exact responsible genetic factors have yet to be identified.

Chung W-H et al, Nature, vol. 428 (6982), p. 486, (2004) and US-A-2005/100926 both disclose that HLA-B1502 is significantly associated with carbamazepine-induced SJS / NET.

These pharmacogenomic studies suggest that the detection of RAF-associated alleles in a patient is a useful approach to assess whether this patient is at risk of developing RAF. This type of molecular diagnosis certified by Clinical Laboratory Improvement Amendments is now offered by reference laboratories in the United States and Europe. 2 ΕΡ2016198Β1

To determine the presence of a particular genetic allele, one or more specific allelic nucleotides need to be detected. In many cases, multiple regions within the allele must be the target for accurate determination. For example, currently available methods for determining an HLA-B allele (e.g., HLA-B * 1502 or HLA-B * 5801) require the detection of at least six regions within that allele.

SUMMARY OF THE INVENTION The present invention provides a method of predicting the risk of a patient developing adverse drug reactions, particularly SSJ, NET or SHS. It has been found that HLA-B * 1502 is associated with SSJ / ΝΕΤ induced by a variety of drugs, for example, carbamazepine (CBZ). In addition, HLA-B * 5801 is particularly associated with allopurinol-induced SSJ / ΝΕ.. HLA-B * 5801 is also associated with allopurinol-induced SHS. Gentle skin reactions induced by CBZ, such as maculopapular cracks, erythema multiforme, urticaria, and fixed drug eruption, are particularly associated with HLA-B * 4601.

Accordingly, the present application provides a method of assessing the risk of a patient according to the claims.

A genetic allele may be detected by the direct detection of regions / nucleotides within the allele using genomic DNA prepared from bio-samples, for example blood, saliva, urine or hair. The allele may also be detected, for example, by serological or microcytotoxicity methods. It can also be determined by detecting an equivalent genetic marker of the 3β2016198Β1 allele, which may be, for example, a PNS (simple nucleotide polymorphism), a microsatellite marker or other types of genetic polymorphisms. In other words, the presence of the haplotype of HLA-B * 1502, 5801 or 4601, rather than the allele itself, is indicative of a risk for adverse drug reactions. Equivalent genetic markers exemplifying the HLA-B B * 1502 haplotype include DRB1 * 1202, Cw * 0801, Cw * 086, A * 1101, and MICA * 019.

Revealed is a method of pharmacogenomic profiling. This method includes determining the presence of at least one HLA-B allele selected from the group consisting of HLA-B * 1502, HLA-B * 5801 and HLA-B * 4601. The presence of at least two alleles selected from the group is determined, such as HLA-B * 1502 and HLA-B * 5801 or the presence of all three alleles is determined. The method may optionally comprise determining other genetic factors. These other genetic factors may be associated with predisposition to any disease or medical condition, including RAF. For example, these other genetic factors may be selected from the group consisting of thiopurine methyltransferase and the genes for long QT syndrome.

Revealed is a method of determining whether a patient is a carrier of HLA-B * 1502 or HLA-B * 5801. This method includes the steps of: (1) detecting a first region selected from HLA-B * 1502 regions 1-6 or HLA-B * 5801 regions 1-6, (2) detecting a second region selected from HLA-B * 1502 regions 1-6 or HLA-B * 5801 regions 1-6, wherein the second region is different from the first region, and (3) determining whether the patient is carrying the allele of interest, in that the presence of the first and second regions indicates that the patient is HLA-B * 1502 or HLA-B * 5801. These regions can be detected by Real Time PCR or ELISA (CSSO-ELISA) ELISA hybridization assay. In one example, the detection of two regions selected from regions 1-6 of HLA-B * 1502 or regions 1-6 of HLA-B * 5801 is sufficient to determine the presence or absence of HLA-B * 1502 or HLA Alternatively, three or more regions within HLA-B * 1502 or HLA-B * 580 are detected. Detection of region 1, region 2, and region 3 of HLA-B * 1502 can be achieved, respectively, by identifying the nucleotides at positions 1 and 3 within region 1, at positions 1 and 6 within region 2 , and at positions 1 and 3 within region 3 (including the nucleotides in the sense strand or at the antisense strand at these positions). Detection of HLA-B * 5801 regions 1-6 can be achieved, respectively, by identifying the nucleotides at positions 1, 2, and 5 within region 1 at positions 1, 4, 6, 7, 8, 15 , 16, and 20 within region 2, at positions 2, 4, 5, 8, and 9 within region 3, at positions 3 and 5 within region 4, at positions 1 and 9 within region 5, and at positions 3 and 10 within region 6.

Revealed is a kit for the detection of a genetic allele, for example, HLA-B * 1502 or HLA-B * 5801. This kit contains a first probe and a second probe, each targeting a region selected from regions 1-6 of HLA-B * 1502 or regions 1-6 of HLA-B * 5801. The two probes target different regions. The kit may also include a third probe targeting an internal control allele. It may also include one or more additional probes for detecting one or more additional regions within HLA-B * 1502 or HLA-B * 5801. 5 ΕΡ2016198Β1

Revealed is a further method of determining whether a compound is a candidate inducing an RAF (e.g., SSJ / NET, SHS, or a milder cutaneous RAF) in a patient bearing an HLA allele (e.g., HLA- B * 1502, HLA-B * 581, or HLA-B * 4601) associated with drug-induced RAF (e.g. CBZ, allopurinol or phenytoin). This method includes the steps of: (1) isolating T cells from an RAF patient bearing an RAF-associated HLA allele, (2) expanding the RAF-inducing drug-reactive T cells, (3) isolating of the antigen presenting cells of the patient, (4) placing the expanded T cells in contact with a compound in the presence of APCs, and (5) examining whether the compound activates the expanded T cells. A T-cell activating compound is a candidate that induces RAF in a patient bearing the same HLA allele.

A patient has a " risk " for an RAF if the probability of the patient developing an RAF is greater than the probability of the general population developing RAF. The likelihood of the patient developing the RAF is at least about 1.3 times greater, more preferably at least about 2 times greater, and still more preferably at least about 3, 4, 5, 6, 7, 8 or 9 times greater, and most preferably at least about 10 times greater as the probability of the general population developing the RAF. The probability may be determined by any method known in the art, such as by using the incidence of risk factors. For example, a particular risk factor is present in 5% of the general population. If this factor is present in 10% of patients having an RAF, then the probability of a patient with this risk factor to develop RAF is 2 fold as high as the probability of the general population to develop RAF. 6 ΕΡ2016198Β1

A " risk factor " for an RAF is a factor that is associated with RAF. The sensitivity of a risk factor is preferably at least about 40%, and more preferably at least about 50%, 60%, 70%, 80%, 85% or 90%. Even more preferably, the sensitivity is at least 95%. The " sensitivity " of a risk factor for predicting RAF is the percentage of RAF patients who have the risk factor. In other words, if each SSJ patient has the A allele, the sensitivity of the A allele to predict SSJ is 100%. If 20 out of 40 SJS patients had the B allele, then the sensitivity of the B allele to predict SSJ is 50%.

An " genetic marker equivalent " of an allele of interest refers to a genetic marker that is attached to the allele of interest, i.e., indicates an imbalance of attachment to the allele of interest. " Pharmacogenomic screening " refers to determining the genetic factors present in an individual that are associated with diseases or medical conditions, particularly adverse drug reactions. Typically, a panel of genetic factors is determined in pharmacogenomic profiling, and the factors may or may not be associated with the same disease, medical condition, or drug reaction.

Details of one or more implementations of the invention are set forth in the following description.

DETAILED DESCRIPTION OF THE INVENTION

Some evidence suggests that the pathogenesis of several RAF's of multiple similar systems involves the MHC-restricted presentation of a drug or its metabolites, which bind directly to MHC molecules or otherwise bind to endogenous proteins, and activation of cells T (Svensson et al., Pharmacol. Rev., 53 (3): 357-379, 2000). CD8 + T cytotoxic T cell infiltration T cells have been found to be dominant in bullous reactions such as SSJ / NET (Hari et al., Clin Exp. Allergy, 31 (9): 1398-1408, 2001), while that CD4 + helper T cells were features of smoother cutaneous RAF, such as maculopapular eruption (Pichler et al., Int., Arch. Allergy Immunol., 113 (1-3): 177-180, 1997).

HLA-B * 1502, HLA-B * 5801, and HLA-B * 4601 were found to be associated respectively with CBZ-induced SSJ / NET, SSP / NET / SHS induced by allopurinol, and with milder cutaneous RAFs (eg, maculopapular rash, erythema multiforme, urticaria, or fixed drug eruption) induced by CBZ. Thus, these HLA-B alleles are useful genetic markers for determining whether a patient is at risk of developing RAF (eg SSJ, NET, SHS, or milder cutaneous RAF) induced by CBZ compounds, allopurinol compounds , or by drugs thereof having similar structures. CBZ, also known as Tegretol, Tegol, G-32883, Biston, Calepsin, Carbatrol, Epitol, Finlepsin, Sirtal, Stazepine, Telesmin, or Timonil, is an aromatic anticonvulsant. Other aromatic anticonvulsants, including phenytoin (Dilantin) and phenobarbital, cause RAF similar to CBZ. Therefore, HLA-B * 1502 may also be used to assess the risk of RAF caused by these other aromatic anticonvulsants. Aromatic anticonvulsants for which HLA-B * 1502 can be used as a risk factor also include compounds or metabolites of CBZ, phenytoin or phenobarbital. The metabolites of these drugs are known in the art (see, for example, Gennis et al., 1991; Leeder, Epilepsy, 39 Suppl 8: S8-16, 1998; Naisbitt et al., Mol. Pharmacol , 63 (3): 732-741, 2003), such as CBZ-10, 11-epoxide, CBZ-10,11-diol, CBZ-2,3-diol, dihydro CBZ, CBZ catechol and CBZ o-quinone , p-hydroxy phenytoin, dihydrodiol phenytoin, catechol phenytoin, methyl catechol phenytoin, and o-quinone phenytoin.

Allopurinol is a drug for hyperuricemia and chronic gout. Table I shows examples of compounds that can induce SSJ / NET in an HLA-B * 1502 carrier.

Table I. SJS-associated drug compounds in HLA-B * 1502 patients

Names of active ingredient or the Trade Name Carbamazepine, Epitol, Tegretol, Microtrol Bipotrol, carbamazepine, carbamazepine, Pharmavene, carbamazepine, Carbatrol, SPD-417 5H-Dibenz [b, f] azepine-5-carboxamide [CAS] Oxcarbazepine, Trileptal GP-47680, GP-47779, GP-47779 MHD, KIN-493, oxacarbazepine, TRI-476, TRI-477, TRI-477 (MHD), Trileptal NP, [CAS]: 5H-Dibenz [b, f] azepine-5-carboxamide, 10,11-dihydro-10-oxo. (S) - (-) - 10-acetoxy-10,11-dihydro-5H-dibenzo [b, f] azepine-5-carboxamide [Î ±] The presence of HLA-B * 5801 in a patient was determined by the method of the present invention, in which the presence of HLA-B * 5801 in the presence of HLA-B * 5801 in a patient indicates that the patient is at risk of SSJ / NET or SHS induced by allopurinol compounds, or compounds structurally similar to allopurinol, or the metabolites thereof. Table II shows examples of the compounds that can induce SSJ / NET or SHS in patients who are HLA-B * 5801 carriers.

Table II: SJS-associated drug compounds in patients who are HLA-B * 5801

Names Structure of the active ingredient Alopurinol, Aloprim, ziloprim, Apo-Allopurinol, Purinol rVj, Oxipurinol, Oxiprim [CAS]: 1H-Pyrazolo [3,4- d] pyrimidine-4,6 (5H, 7H) -dione Febuxostat, TEI-6720, TMX-67 [CAS]: 5-thiazolecarboxylic acid, 2- [3-cyano-4- (2-methylpropoxy) phenyl] -4-methyl- " " And a " " Y-700 1- (3-Cyano-4-neopentyloxyphenyl) pyrazole-4-carboxylic acid V .. V ^ V1 '" v Λ 10 Ρ 2016198Β1 modafinil, Sparlon (New Drug Formulation), [CAS]: Acetamide, 2- [ The presence of HLA-B * 4601 in a patient indicates that the patient is at risk of developing mild cutaneous RAF, for example CBZ-induced maculopapular rash.

Other HLA-B alleles may also be predisposed for cutaneous RAF. For example, ankylosing spondylitis is strongly associated with HLA-B27 alleles, such as B * 2701-b * 2723. (Khan, Curr. Opin Rheumatol., 12 (4): 235-238, 2000 / Feltkamp et al., Curr. Opin. Rheumatol., 13 (4): 285-290, 2001). The presence of a genetic marker (e.g., an HLA allele) may be determined by the direct detection of that marker or the particular regions within it. Genomic DNAs for allele detection can be prepared from a patient by methods well known in the art, for example, the PUREGENE DNA purification system from Gentra Systems, Minnesota. Detection of a region within a genetic marker of interest includes examination of the nucleotide (s) located in the sense or antisense strand within that region. Methods known in the state of the art can be used to detect a particular region, for example, sequence specific oligonucleotide hybridization, real-time PCR, or CSSO-ELISA (M. Hiratsuka et al, J. of Biochemical and Biophysic. Methods, 67: 87-94, 2006). The presence of HLA-B * 1502 can be determined by detecting at least two of regions 1 to 6 shown in Figure 1. The presence of these regions can be determined by detecting nucleotides at certain positions within these regions, e.g. positions 1 and 3 in region 1, positions 1 and 6 in region 2, and positions 1 and 3 in region 3. The presence of any two of regions 1 to 6 indicates that the patient is a carrier of HLA-B * 1502 . The presence of HLA-B * 5801 can be determined by detecting at least two of regions 1 to 6 shown in Figure 2. The presence of these regions can be determined by detecting nucleotides at certain positions within these regions, e.g., positions 1 2, and 5 in region 1, positions 1, 4, 6, 7, 8, 15, 16, and 20 in region 2, positions 2, 4, 5, 8, and 9 in region 3, positions 3 and 5 in region 4, positions 1 and 9 in region 5, and positions 3 and 10 in region 6. The presence of any two of regions 1 to 6 indicates that the patient is a carrier of HLA-B * 5801.

DNA products obtained from PCR can be detected using sequence specific probes, for example, TaqMan hydrolysis probes, Beacons, Scorpions; or hybridization probes. These probes are designed in a manner such that they bind to the regions of interest, for example, regions 1 to 6 of HLA-B * 1502 or regions 1 to 6 of HLA-B * 5801. PCR products can also be detected by DNA binding agents, for example, SIBR® Green. The presence of an allele of interest can also be determined by detecting the equivalent genetic markers for the allele. Genetic markers near the allele of interest tend to co-segregate, or show a binding imbalance, with the allele. Accordingly, the presence of these markers (genetic markers equivalent to ε2020198Β1) is indicative of the presence of the allele of interest, which, in turn, is indicative of a risk for the development of RAF. Exemplary genetic markers equivalent to HLA-B * 1502 include DRB1 * 12 02, Cw * 0801, Cw * 0806, A * 1101, and MICA * 019. Exemplary HLA-B * 5801 exemplary labels include A * 3303, Cw * 0302, DRB1 * 0301, and MICA * 00201.

An equivalent genetic marker may be of any type, for example, an HLA allele, a microsatellite marker, or a single nucleotide polymorphism marker (PNS). A useful equivalent genetic marker is usually about 200 kb or less (e.g., 100 kb, 80 kb, 60 kb, 40 kb, or 20 kb) of the allele of interest. The methods described above or known in the prior art may be used to detect the presence or absence of an equivalent genetic marker.

Alternatively or in addition, RNA, DNA, or protein products of the alleles of interest may be detected to determine the presence or absence of the allele. For example, serotyping or microcytotoxicity methods may be used to determine the protein product of an HLA allele.

To further enhance the accuracy of the prediction of risk, the allele of interest and / or its equivalent genetic marker can be determined together with the genetic markers of the accessory molecules and the co-stimulatory molecules that are involved in the interaction between antigen presenting cells and T cells. These genetic markers include microsatellite markers and single nucleotide polymorphism (PNS) markers. The accessory and costimulatory molecules include cell surface molecules (eg, CD80, CD86, CD28, CD4, CD8, T cell receptor (TCR), ICAM 1, CDlla, CD58, CD2, etc.) and inflammatory or proinflammatory cytokines, chemokines (e.g., TNF-α), and mediators (e.g., complements, apoptosis proteins, enzymes, extracellular matrix components, etc.). Also of interest are genetic markers of drug metabolizing enzymes which are involved in the bioactivation or detoxification of the drugs. These genetic markers also include the microsatellite and PNS markers. The drug metabolizing enzyme includes phase I enzymes (e.g., the cytochrome P450 superfamily, etc.) and phase II enzymes (e.g., microsomal epoxide hydrolase, arylamine N-acetyltransferase, UDP-glucuronosyl transferase, etc.). ).

Revealed is a method for pharmacogenomic profiling. A panel of genetic factors is determined for a particular individual, and each genetic factor is associated with predisposition to a disease or medical condition, including RAF. In the method, the panel of genetic factors includes at least one allele selected from the group consisting of HLA-B * 1502, HLA-B * 5801 and HLA-B * 4601. The panel may include two alleles or all three alleles of the group. In addition to HLA-B * 1502, 5801 and / or 4601, the panel may include any other known genetic factors, such as thiopurine methyltransferase and the genes for long QT syndrome. The genetic markers for accessory molecules, costimulatory molecules and / or drug metabolizing enzymes described above may also be included.

Revealed is a kit containing probe for detecting genetic markers, for example, HLA-B * 1502, HLA-B * 5801 or HLA-B * 4601. The term " probe " used herein refers to 14 ΕΡ2016198Β1 any substance useful for detecting another substance. Thus, a probe may be an oligonucleotide or a conjugated oligonucleotide that specifically hybridizes to a particular region within an allele of interest. The conjugated oligonucleotide refers to an oligonucleotide covalently linked to the chromophore or to molecules containing a ligand (e.g., an antigen), which is highly specific to a molecular receptor (e.g., an antigen-specific antibody). The probe is also a PCR primer, along with another primer, to amplify a particular region within the allele of interest. In addition, the probe may be an antibody that recognizes an allele of interest or an allele protein product. Optionally, the kit may contain a probe targeting an internal control allele, which may be any allele presented in the general population, for example GAPDH, β-actin, KIR. The detection of an internal control allele is intended to ensure the performance of the kit. The kit may also include tools and / or reagents for collecting biological samples from patients as well as those for the preparation of the genomic DNA, cDNA, RNA or proteins from the samples. For example, PCR primers for amplifying relevant regions of genomic DNA may be included. The kit may also contain probes for genetic factors useful in pharmacogenomic profiling, for example, thiopurine methyltransferase. The kit may contain a first probe and a second probe, each for detecting a region selected from regions 1 to 6 of HLA-B * 1502 or regions 1 to 6 of HLA-B * 5801. second probes target different regions. These two probes may be a pair of PCR primers, or tagged oligonucleotides useful in ηΡ2016198Β1 in hybridization assays. Optionally, the kit may include a third probe for detecting an internal control allele. It may also include additional probes for detecting additional regions within HLA-B * 1502 or HLA-B * 5,801.

Revealed is a method of identifying a drug compound that induces an RAF (e.g., SSJ / NET or SHS) in a patient bearing the RAF-associated HLA allele (e.g., HLA-B * 1502, HLA-B * 5801 or HLA-B * 46 01). It is suggested that the drugs may be presented by certain HLA complexes to activate the T lymphocytes, consequently inducing RAFs. It is suggested that T cells reactive to these drugs are involved in the development of RAF induced by these drugs. Thus, compounds that can activate these T cells are candidates to induce RAF in a patient bearing one or more HLA alleles associated with these RAFs. As a result, this method can be used as a selection method in the development of new drugs to find the drug compounds that could induce such RAFs. Genotyping can be performed in an RAF patient to determine if the patient is carrying an HLA allele associated with the disease. T lymphocytes and antigen presenting cells (e.g., B cells or monocytes) can be isolated from the patient and cultured in vitro by following methods well known in the art. (Naisbitt DJ, Mol. Pharmacol 2003 Mar; 63 (3): 732-41, Wu et al, J. Allergy Clin Immunol 2006 Jul; 118 (1): 233-41. E-published 27 April 2006 ). B cells isolated in this manner can be transformed by the Epstein-Bar virus to generate B cell lines. Drug reactive T cells can be expanded in the presence of the drug and autologous antigen presenting cells. The expanded T cells can then be exposed to a test compound in the presence of autologous antigen presenting cells to determine whether the test compound can activate the T cells. If so, the test compound is a candidate that can induce RAF in a patient bearing the same HLA allele.

The following examples are to be construed as merely illustrative, and not limiting the remainder of the disclosure in any manner. EXAMPLE 1. Detection of HLA-B * 1502 and HLA-B * 5801 Using Real-Time PCR

Genomic DNAs were extracted from a patient's blood or saliva. Three pairs of PCR primers from each of regions 1 and 5, regions 1 and 4, or regions 1, 3 and 4 targeted HLA-B * 1502 (see Figure 1) were synthesized. In addition, a pair of primers targeting HLA-B * 5801 regions 2 and 4 (see Figure 2) was synthesized. The target regions were amplified and detected using the SIBR® Green Real Time PCR system (Applied Biosystem). Briefly, primers, genomic DNAs and the master SIBR® Green PCR mix (included in the real-time PCR system) were mixed together and the PCR was performed by: (i) activating the polymerase at 95 ° C for ten (ii) denaturation at 95 ° C for fifteen seconds and annealing / elongation of DNA strands at 71 ° C for one minute, (iii) execution of 40 denaturation / annealing / elongation cycles, and (iv) product dissociation amplified from its template at 95 ° C for fifteen seconds, and at 60 ° C for one minute. The PCR amplification of a γ2016198Β1 cell-killing immunoglobulin-like receptor (KIR) was applied as an internal control. The presence or absence of HLA-B * 1502 or HLA-B * 5801 in a patient was determined on the basis of the Ct value of HLA-B * 1502 or HLA-B * 5801 and the difference in Ct values ACt) between HLA-B * 1502 / HLA-B * 5801 and KIR. The Ct value (limit cycle) is determined by identifying the number of the cycle in which the intensity of the reporter dye emission is above the background noise. The limit cycle is determined at the most exponential phase of the reaction and is more reliable than the endpoint measurements of the cumulative PCR products used by traditional PCR methods. The limit cycle is inversely proportional to the number of copies of the target mold, the larger the mold number, the lower the measured limit cycle. 170 samples of genomic DNA extracted from human B cell lines and 35 samples of genomic DNA prepared from human blood or human saliva were tested for the presence of HLA-B * 1502 following the method described above. The Ct values of HLA-B * 1502 and KIR were in the range of 23-27 and 19-25, respectively. HLA-B * 1502 were recognized as positive when the AC t interval between HLA-B * 1502 and KIR was less than 7. In these supercontrol vectors, 15 g of HLA-B * 1502 DNA were present and were verified by kits Dynal SSO, and the results indicate that the sensitivity and specificity of this method reach > 99%. 170 samples of genomic DNA extracted from human B cell lines and 87 samples of genomic DNA prepared from human blood or human saliva were tested for the presence of HLA-B * 5801 following the method described above. For DNA samples derived from HLA-B * 5801-positive patients, the Ct values of 18 ΕΡ2016198Β1 HLA-B * 5801 and KIR were in the range of 22-28 and 10-26, respectively. HLA-B * 5801 were recognized as positive when the ACT interval between HLA-B * 5801 and KIR was less than 7. For DNA samples derived from HLA-B * 5801 negative patients, the Ct value of HLA -B * 5801 was approximately 34 and ACt was greater than 13. In all samples, 51 g of 51 DNA were found to be HLA-B * 5801 positive, and were verified by Dynal SSO kits. These results indicate that the sensitivity and specificity reached > 99%.

EXAMPLE 2. Detection of HLA-B * 1502 and HLA-B * 5801 Through the Use of CSSO-ELISA

The procedures for performing CSSO-ELISA are outlined in Figure 3.

Generally, when using genomic DNA as templates, the PCRs were run to obtain products containing the specific regions shown in Figure 1 or Figure 2. Both the forward primer and the reverse primer were labeled with a Ligand 1 (Ll ), which could be recognized by Molecular I linked with an enzyme (e.g., HRP). PCR reactions were designed and performed to obtain products containing one or more specific regions, i.e. HLA-B * 1502 regions 1 to 6 or regions 1 to 6 of HLA-B * 5801. Competition sequence-specific oligonucleotides (CSS01 and CSS02) were designed. CSS01 specifically recognizes one of the regions 6a of HLA-B * 1502 or regions 1 to 6 of HLA-B * 5801, and CSS02 was designed to be the common type primer of HLA-B * 15 (i.e., non-alleles -B * 1502 or non-B * -5801. CSS01 was labeled with Ligand 2, which can be recognized by II Molecular coated on a reaction plate or strip. The PCR products thus obtained were hybridized with the two After the removal of any free molecule by washing, the substrate of the enzyme was added to the reaction plate The enzymatic reaction, indicated by the presence of a color, is indicative of the presence of HLA- B * 1502 or HLA-B * 5801. EXAMPLE 3. Correlation between SSB / NET induced by CBZ and HLA-B * 1502

A total of 238 RAF patients (descendants of Mongoloides or Mongoloides) were recruited from the Chang Gung Memorial Hospital or from several other medical centers throughout Taiwan for this study. Its history of drug ingestion including dosage and duration, and RAF phenotypes were recorded. Diagnostic criteria for clinical morphology were defined according to Roujeau, J. Invest Dermatol., 102 (6): 28s-30s, 1994. For example, SJS was defined as the skin detachment of less than 10% of the area of the body surface, SSJ-NET overlap as a skin detachment of 10-30%, and NET as more than 30%. The SSJ, the SSJ-NET and NET overlap are collectively referred to as SSJ / NET.

For each patient, the suspected drug was withdrawn and the patient observed for symptoms. Patients who developed a cutaneous RAF that did not give in on drug withdrawal were excluded.

According to the criteria described above, 112 patients were diagnosed with SSJ / NET and 126 patients had a milder hypersensitivity reaction to several drugs. Among the 112 SJS / NET patients, 42 were exposed to CBZ (tegretol), 17 had ingested allopurinol, and 53 were on medication with 20 ΕΡ2016198Β1 exception of CBZ and allopurinol. 73 patients tolerant to tegretol were included as controls. Volunteers from the general population of Taiwan (n = 94; age range: 20 to 80 years) were also recruited. The study was approved by the institutional review board, and informed consent was obtained.

All these patients were submitted to genotyping. Briefly, reagents for performing a reverse-line transfer assay using sequence-specific oligonucleotide (SSO) were purchased from DYNAL Biotech Ltd. (Bromborough, UK). PCR products were generated by using biotinylated primers for the second and third exons of HLA class I or class II loci, and then hybridized to an SSO line transfer of probes immobilized on a nylon membrane. The presence of biotinylated PCR product bound to a specific probe is detected using the strong root streptavidin peroxidase (HRP) and a soluble chromogenic substrate to produce a " blue line " position of the positive probe. The probe reactivity pattern was interpreted by Dynal RELI ™ SSO genotyping software (DYNAL Biotech Ltd, Bromborough, UK). Potential ambiguities have also been resolved by sequence-based typing and DNA sequencing performed according to the IHWG (International Histocompatibility Working Group) technical manual, see Genomic Analysis of the Human MHC DNABased Typing for HLA Alleles and Linked Polymorphisms. Mareei G.J. Tilanus, editor in chief, ISBN No. 0-945278-02-0.

For some patients, the genotyping of PNS was performed using the high throughput MALDI-TOF mass spectrometry. Briefly, primers and probes were designed using the SpectroDESIGNER software (Sequenom, San Diego, CA, USA). Multiplex polymerase chain reactions (PCR) were performed, unincorporated dNTPs were dephosphorylated using shrimp alkaline phosphatase (Hoffman-LaRoche, Basel, Switzerland), followed by primer extension. The purified primer initiation reaction was stained on a 384-element silicon chip (SpectroCHIP, Sequenom), analyzed by using a Spectrum READER (Sequenom) Bruker Biflex III mass spectrometer MALDI-TOF SpectroReader (Sequenom) and spectra processed with SpectroTYPER (Sequenom) .

The allele frequencies in the different groups were compared by the Chi squares method with the Yates correction by constructing 2 x 2 frames. The P values were corrected for the comparisons of the multiple HLA alleles (Pe) by multiplying the values of P by the observed number of HLA alleles present within the loci. The odds ratios were calculated with Haldane's modification, which adds 0.5 to all cells to accommodate possible counts equal to zero.

As shown in Table 1, a DNA variant allele at the HLA-B locus (HLA-B * 1502) was associated in patients with drug-induced SSJ / NET, particularly in patients receiving CBZ (tegretol). 22 ΕΡ2016198Β1

Table 1. Frequency of HLA-B * 1502 in 42 Taiwanese patients having CBZ_ induced SSJ-NET

Allele Patients N = 42 Controls1 N = 142 Controls2b N = 94 Controls3c N = 73 X2 Probability Ratio Pa B * 1502 B * 1502 42 (100%) 42 (100%) 9 (6.3)% 5 (5.3 %) 137.28 110.919 1194.47 1383.2 3, 6x10-3 ° 2.15x10 -24 B * 1502 42 (100%) 3 (4.1%) 98.936 1712.9, 1x10 " 22a, patients who had a smoother RAF b, Taiwanese general population c, patients who are tolerant to CBZ X2, Chi-squared with Yates Pc correction, calculated by multiplying the observed values of the HLA-B (35) alleles. with the exception of SSJ of crude P by the number 0 HLA-B * 1502 was detected in 42 of 42 (100%) SSJ / NET patients who received CBZ. The allele was also found in 17 of 53 (32%) SSJ / NET patients who received other drugs (8 phenytoin, 2 allopurinol, 2 amoxicillin, 1 sulfasalazine, 1 ketoprofen, 1 ibuprofen, and 2 unknown drugs). In particular, 8 out of 17 patients (47.05%) who developed SSJ / NET after phenytoin ingestion were also carriers of the HLA-B * 1502 allele. On the other hand, the allele was found only in 4.1% (3/73) of the CBZ tolerant group, in 0% (0/32) of the phenytoin tolerant group, in 6.3% (9/142) of the patients who had smoother ANS except SJS, and in 5.3% (5/94) of the general population. Through the use of the tolerant group as a control, the ratio of the probabilities, sensitivity, specificity, positive predictive value, and negative predictive value for CBZ-induced B * 1502 associated with SJS / NET was 171%, 100%, 95, 89%, 96.0%, and 100%, respectively. With such a high predictive value and sensitivity, typing of this HLA-B allele can be used in the identification of patients at high risk for drug-induced SJS / NET, particularly CBZ or phenytoin-induced SSJ / NET. 23 ΕΡ2016198Β1

It has been found that CBZ-induced soft RAFs are associated with another allele, HLA-B * 4601. Ten of the sixteen (62.5%) patients with these milder CBZ reactions had HLA-B * 4601. On the other hand, the allele was found only in 26% (19/73) of the CBZ tolerant group. The ratio of the probabilities for CBZ-induced milder skin RAF associated with B * 4601 was 4.73. Consequently, HLA-B * 4601 can be used in the risk assessment for mild cutaneous RAF induced by CBZ.

Table 2. Phenotype / genotype data of patients having CBZ_ ID-induced skin RNA ID Suspected drug Phenotype HLA-B genotype 1 Carbamazepine SSJ B * 1502 / * 3802 2 Carbamazepine SSJ B * 1502 / B * 3501 3 Carbamazepine SSJ B * 1502 / B * 4006 4 Carbamazepine SSJ B * 1502 / B * 3802 5 Carbamazepine SSJ B * 1502 / B * 3802 6 carbamazepine, phenytoin SSJ B * 1502 / B * 3802 Carbamazepine SSJ B * B Carbamazepine SSJ B * 1502 / B * 5801 10 Carbamazepine SSJ B * 1502 / B * 5801 11 Carbamazepine SSJ B * 1502 / B * 1525 12 Carbamazepine SSJ B * 1502 / B * B * 1502 / B * 4006 Carbamazepine SSJ B * 1502 / B * 5801 15 Carbamazepine Overlap SSJ / NET B * 1301 / B * 1502 16 Carbamazepine Overlap SSJ / NET B * 1502 / B * 3501 17 Carbamazepine SSJ B * 1502 / B * 3802 18 Carbamazepine SSJ B * 1502 / B * 4601 19 Carbamazepine SSJ B * 1301 / B * 1502 24 ΕΡ2016198Β1 20 Carbamazepine SSJ B * 1502 / B * 5801 21 Carbamazepine SSJ B * 1502 / B * 4601 Carbamazepine, ΑΙΝΕ SSJ B * 15 02 23 Carbamazepine SSJ B * 1502 / B * 3501 24 Carbamazepine SSJ B * 1502 / B * 4601 25 Carbamazepine SSJ B * 502 / B * 4601 26 Carbamazepine SSJ B * 1502 / B * 5801 Carbamazepine SSJ B * 1502 / B * 4001 29 Carbamazepine SSJ B * 15 02 30 carbamazepine, meloxicam, sulidan, phenytoin SSJ B * 1502 / B * 5801 Carbamazepine SSJ B * 1502/4601 32 Carbamazepine SSJ B * 1502/5801 33 Carbamazepine SSB B * 1502/4601 34 Carbamazepine SSJ B * 1502/5502 35 Carbamazepine SSJ B * 15 02 36 carbamazepine, phenytoin SSJ B * 1502/4002 37 Carbamazepine SSJ B * 1502 / SSB B * 1502 40 Carbamazepine Overlap SSJ / NET B * 1502/4001 41 Carbamazepine Overlap SSJ / NET B * 1502/4601 42 Carbamazepine SSJ B * 1502/3802 43 Carbamazepine maculopapular eruption B * 5801 / B * 4601 44 Carbamazepine erythema multiforme B * 400 / B * 4601 45 Carbamazepine eruption maculopapular B * 1301 / B * 4001 46 Carbamazepine angioedema EB * 4601 / B * 5401 47 Carbamazepine eruption maculopapular B * 4001 / B * 4601 48 Carbamazepine, ΑΙΝΕ maculopapular eruption B * 4001 / B * 4001 49 Carbamazepine maculopapular eruption B * 1301 / B * 5502 50 Carbamazepine swelling of the lips, oral and genital ulcer B * 4601 / B * 5801 51 Carbamazepine maculopapular eruption B * 4001 / B * 5101 54 Carbamazepine maculopapular eruption B * 1301/4001 53 Carbamazepine maculopapular eruption B * 4001 / B * 4601 56 Carbamazepine maculopapular eruption B * 4001 / B * 5101 54 Carbamazepine angioedema EB * erythema multiforme B * 4601 / B * 5401 57 Carbamazepine maculopapular rash B * 4601 58 Carbamazepine erythema multiforme B * 4601/5101 EXAMPLE 4. Correlation between Allopurinol-Induced and HLA-B * 58 01

It has been found that HLA-B * 5801 is associated with allopurinol-induced SSJ / NET. This allele of HLA-B was found in all 17 (100%) severe SSJ / RAF patients treated with allopurinol (Tables 3 and 4) but were found in only 18% of the general population of Taiwan (odds ratio 155 , sensitivity of 100%, specificity of 82%, positive predictive value of 84.7%, negative predictive value of 100%, Pc = 3.7 x 1CT9). These results suggest that HLA-B * 5801 is a useful genetic marker, either alone or in combination with other genetic markers, to assess whether a patient on allopurinol is at risk of developing SSJ / NET.

Table 3. HLA-B * 5801 frequency in 17 Taiwanese patients with severe cutaneous RAF induced by allopurinol

Allele Patients Controls3 Controls2b X2 Pc ratio n = 17 n = 142 n = 94 Odds

(100%) 17 (18.0%) 41.7 15.5 - 3.7 x 10 9 a, b) 5801 17 (100%) 26 (18.3%) 47.2 153, 86 2,1X10 " patients who had an RAF with the exception of cutaneous RAF induced by allopurinol b, general population of Taiwan c, patients who are tolerant to CBZ X2, Chi-squared with correction of Yates Pc, calculated by multiplying the values of gross P by the observed number of the HLA-B alleles (35). 26 ΕΡ2016198Β1

Table 4. Phenotype / genotype data of patients with allopurinol-induced cutaneous RAF Patient ID Suspected drug Phenotype HLA-B genotype 59 allopurinol SSJ B * 0705 / B * 5801 60 allopurinol SSJ B * 4 0 01 / B * 5 8 Allopurinol SSJ B * 5801 64 allopurinol SSJ B * 5751 62 allopurinol SSJ B * 5801 6 allopurinol SSJ B * 5751 6 allopurinol SSJ B * 3901 / B * 5801 allopurinol SSJ B * SSJ B * 4 0 01 / B * 5 8 01 67 allopurinol SSJ B * 1502 / B * 5801 68 allopurinol SSJ B * 40 01 / B * 5 8 01 69 allopurinol SSJ and vasculitis in leg B * 4601 / B * 5801 70 allopurinol SSJ and lichenoid B * 4001 / B * 5801 71 allopurinol SSJ B * 4002 / B * 5801 72 allopurinol SSJ B * 4001 / B * 5801 73 allopurinol SSJ B * 5101 / B * 5801 74 allopurinol SSJ B * 1301/5801 75 allopurinol SSJ B * 5801 EXAMPLE 5. Correlation between HLA-B * 5801 and SHS induced by Allopurinol

HLA-B * 5801 has also been found to be linked to allopurinol-induced SHS, which includes rash (eg diffuse macuopapular, exfoliative dermatitis), fever, eosinophilia, atypical circulating lymphocytes, leukocytosis, acute hepatocellular injury, or worsening of renal function (Arellano et al., Ann. Pharmacoter., 27: 337, 1993). 31 patients were studied, among which 21 had SJS, 3 had SJS / NET, 1 had NET, and 15 had SHS. In all reported cases, allopurinol was considered to be the offending drug if the occurrence of RAF syndrome occurred within the first two months of exposure to allopurinol and symptoms of RAF disappeared upon withdrawal of the drug. Patients with any of the following conditions were excluded: absence of symptoms after re-exposure to allopurinol, and patients with milder eruptions of the skin that did not meet SHS, SSJ or NET criteria. The appearance of SHS symptoms for all 31 patients occurred within the first two months of exposure to allopurinol and two patients had a second attack within two days of re-exposure to allopurinol. Twelve patients received other drug (s) in addition to allopurinol, but their medical records did not reveal any RAF when these drugs were ingested concomitantly without allopurinol. All patients had hyperuricemia and / or gout arthritis, as well as other chronic diseases, including hypertension (14/31), chronic kidney disease (16/31), and diabetes (9/31).

Ninety-eight gout arthritis patients who had ingested allopurinol for at least six months (mean = 38 months, range = 6-107 months) without any RAF syndrome were included as allopurinol tolerant control. The gender distribution of the tolerant group is comparable to the general prevalence of gout in Chinese people. In addition, 93 normal subjects served as the normal control group. The demographic variables of these three groups are shown in Table 5. 28 ΕΡ2016198Β1

Table 5. Demographic variables, dosage and duration of exposure to allopurinol in patients with severe RAF in tolerant patients as well as in normal RAF subjects Tolerant Individuals (n = 31) (n = 98) normal (n = 93) Male Sex 12 89 52 Female 19 9 41 Age (years) Mean (range) 57.9 (18-91) 57.3 (21-84) 53.9 (22-91) Alupurinol dosage (mg / day) Mean ) 143.3 (50-300) 159.2 (100-400) None Duration of exposure to allopurinol Mean (range) 28.2 days 38 months (6 - 107) None (1 - 56) 0 HLA-B allele * 5801 was present in all 31 (100%) patients with severe allopurinol-induced RAF in 16 (16.3%) of the 98 allopurinol tolerant patients (315 Pc &lt; 10-15), and in 19 (20%) of the 93 normal subjects (odds ratio 241, Pc <10 "13). In relation to the allopurinol tolerant group, the absence of HLA-B * 5801 had a 100% negative predictive value for allopurinol-induced RAF, and the presence of this allele had a positive predictive value of 66%.

Consequently, HLA-B * 5801 is a useful marker with high specificity (84%) and sensitivity (100%) for severe RAF induced by allopurinol, including cutaneous RAFs (eg SSJ / NET or SHS) and DRESS drug with eosinophilia and systemic symptoms) induced by allopurinol. EXAMPLE 6. Genetic markers equivalent to HLA-B * 1502 or HLA-B * 5801

Genetic markers near an HLA allele of interest tend to co-segregate, or show a binding ÎΡ2016198Β1 imbalance, with the allele of interest. As a consequence, the presence of these markers (equivalent genetic markers) is indicative of the presence of the allele.

To test the incidence of potential equivalent genetic markers in RAF patients, several markers in the HLA-B * 1502 haplotype were determined for their association with RAF. HLA-B * 15β haplotype HLA markers, such as DRB1 * 1202, Cw * 0801, Cw * 0806, A * 1101, and MICA * 019, certainly had a significantly high frequency in SJS / NET patients who had been exposed to CBZ (Table 6).

The markers associated with HLA-B * 5801 were also determined. The allele distribution was analyzed in four patients who were homozygous for HLA-B * 5801 and the ancestral haplotype of this allele was defined as including HLA-A * 3303, Cw * 0302, B * 5801 and DRB 1 * 0301. This ancestral haplotype was present in 12 (38.7%) of the 31 patients of allopurinol RAF (Table 7), but only in 7.1% of tolerant patients and in 9.7% of normal individuals.

Table 6. Correlation between ancestral haplotype markers of B * 1502 and RAF CBZ SSJ / NET (n = 42) CBZ tolerant (n = 16) CBZ tolerant (n = 73) Allopurinol SSJ / NET HLA-B * 1502 42 (100%) 0 (0%) 3 (4.1%) 1 (5.8%) 5 (5.3%) HLA-Cw * 0 8 01 38 (90%) ND 10 (13.7%) 2 (11.7%) 10 (10.6%) HLA-Cw * 0 8 0 6 3 (7.1%) ND 0 (0%) 0 (0 HLA-DRB1 * 12 01 35 (83.3%) ND ND ND 19 (20.2%) HLA-A * 1101 31 (73.8%) ND ND ND 28 (29.8% %) 30 ΕΡ2016198Β1

Table 7. Single or combined HLA-B * 5801 haplotype locus frequencies in patients with severe allopurinol-induced RAF in patients tolerant to allopurinol and in normal individuals

Allopurinol- (n = 31) RAF Tolerant to Alupurinol (n = 98) Normal subjects (n = 93) B * 5801 31 (100%) 16 (16.3%) 19 (20.4%) 2 Cw * 0.302 (21.5%) 21 (93.5%) 1.5 (17.3%) 19 (20.4%) A * 3303 20 (64.5%) 18 (18.4%) 20 (21.5%) DRBI * 0301 21 (67.7%) 14 (14.3%) 14 (15.1%) B * 5801, Cw * 0302 29 (93.5%) 15 (15.3%) 19 (20.4%) B (9.3%) 16 (17.2%) A * 3303 B * 5801, Cw * 0302, 19 (61.3%) 9 (9.3% 2%) 10 (10.8%) DRB1 * 0301 B * 5801, Cw * 0302, 12 (38.7%) 7 (7.1%) 9 (9.7% A * 3303, DRB1 * 0301 1 Relation (Allopurinol-RAF / Tolerant): 315 (95% CI, 18.3-5409.5), pc = 7.5 x 10 -16. (95 &quot; Cl, 14.1-4111), Po = 6.1 x 10 -13.

MHC markers associated with HLA-B * 5801 were also determined using the short tandem repeat polymorphism (PRTC) assay. Briefly, twenty highly polymorphic satellite markers located in the MHC region were selected from the NCBI database (i.e., D6S258, D6S2972, D6S510, D6S265, D6S388, D6S2814, HLAC_CA1, HLABC_CA2, MIB, MICA, TNFd, BAT2_CA, D6S273, D6S1615 , DQCAR, G51152, D6S2414, D6S1867, D6S1560, and D6S1583). The mean heterozygosity of these markers was 0.72 with an estimated spacing of 230 kb.

The primers were designed based on the sequences of these labels described in the database. PCRs were performed to amplify and detect the presence or absence of these markers in the Î »2016198 1 patients by using GeneAmp 9700 thermocycles (Applied Biosystems, Foster City, CA, USA) (in a 5 μ volume volume containing 10 ng genomic DNA and 0.33 μg of each primer). Up to six PCR products having appropriate sizes and exhibiting fluorescent signals were pooled prior to capillary gel electrophoresis. The size of the polymorphic amplicons was determined by the ABI 3730 DNA sequencer (Applied Biosystems) electrophoresis using the size standard LIZ500 as an internal size standard (Applied Biosystems). Allele sizing was calculated using version 3.0 of the GENMAPPER program (Applied Biosystems). The call and deposit of the alleles were performed using the SAS program. Three subjects from the CEPH control (1331-01, 1331-02, 1347-2) and H20 were included in all genotyping experiments for quality control purposes.

An allele block located between HLA-C and TNFÎ ± was found in the allopurinol-induced RAF patient group, but not in the allopurinol tolerant group, using a binding imbalance plot. In this block, a haplotype (MIB * 358-MICA * 206-TNFd * 140) near the HLA-B allele was identified. The association of this haplotype with the RAFs is consistent with the association of HLA-B * 5801 with the same RAFs (p = 0.0018). When using PRTC markers and sequencing of the MICA allele, all allopurinol-induced RAF patients were found to be carriers of the same B allele (B * 5801), the MICA allele (MICA * 00201), and the TNF-PRTC marker (TNFD * 140). Except for one patient, all others were also found to carry the same MIB marker (MIB * 358). 32 ΕΡ2016198Β1 EXAMPLE 7. Cross Reactivity of Reactive T Cells to CBZ, Oxcarbazepine and Licarbazepine

Two patients who had CBZ-induced SSJ / NET were recruited at the Chang Gung Memorial Hospital. One of the patients was HLA-B * 1502 / b * 4601, and the other of HLA-B * 1502 / b * 5101. Genomic DNAs were extracted from patients using the PUREGENE DNA purification system (Gentra Systems, Minnesota, USA). HLA-B alleles were verified by the use of sequence-specific oligonucleotide reverse transcripts (DYNAL Biotech Ltd., Bromborough, UK).

Peripheral blood mononuclear cells (PBMC) were isolated from patients by Ficoll-Isopaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ). A part of PBMCs was transformed by the Epstein-Bar virus to establish autologous B-cell lines.

CBZ-reactive T cells were expanded as described below. Patient prepared PBMCs were cultured in a complete RPMI medium containing 10% heat-deactivated human serum, IL-2 (25 U / ml), and CBZ (25æg / ml) (Sigma) in a 5% incubator of C02 cells at 37 ° C for seven days T cells were then expanded by co-cultivation with irradiated autologous B cells (50 Gy) in the presence of CBZ for ten days.After two cycles of the above co-cultivation procedure, the cells T activated by CBZ were collected and submitted to ELISPOT (eBioscience) assays.

CBZ-reactive T cells were tested for their cross-reactivity compounds, for example CBZ 10, 33 ΕΡ2016198Β1 11-epoxide, Oxcarbazepine (brand: trileptal),

Licarbazepine, and sunlindac. Briefly, T lymphocytes (5 x 103 cells) were mixed with autologous B cells (5 x 10 4 cells) in 200 μl of RPMI medium containing 10% FBS in the presence or absence of a test compound. The cells were then incubated for 24 hours in the wells of an ELISPOT plate coated with anti-interferon γ (Millipore) antibodies. After incubation, the cell culture supernatant was harvested and the γ-interferon contained therein was detected using the antibody-mediated methods known in the art.

The results of this study indicate that CBZ-reactive T cells were retro-reactive to CBZ 10, 11-epoxide, Oxcarbazepine, and Licarbazepine, but not to Sulindac.

Lisbon, July 23, 2013 34

Claims (9)

A method of evaluating a risk of a human patient to develop an adverse drug reaction in response to a drug, comprising detecting the presence of HLA-B * 1502 in a sample obtained from the patient, and correlating the presence of HLA-B * 1502 in the sample at an increased risk for an adverse drug reaction in the patient in response to the drug, wherein the adverse drug reaction is Stevens-Johnson syndrome or toxic epidermal necrolysis and the drug is oxcarbazepine or licarbazepine. The method according to claim 1, wherein the drug is oxcarbazepine. The method according to claim 1, wherein the drug is licarbazepine. The method of any of claims 1-3, wherein the sample obtained from the patient is a DNA sample. The method of any of claims 1-4, wherein the presence of the HLA allele is determined by hybridization with an oligonucleotide that specifically hybridizes to the allele. The method of any of claims 1-3, wherein the sample obtained from the patient is an RNA sample or a protein sample. The method of any of claims 1-5, wherein the sample is obtained from peripheral blood, saliva, urine, hair of the patient. The method of any of claims 1-7, wherein the adverse drug reaction is Stevens-Johnson syndrome. The method of any of claims 1-8, wherein the adverse drug reaction is toxic epidermal necrolysis. or to the Lisbon, July 23, 2013 2